We have conducted two studies to assess how currently available treatments for HCV affect the response of innate immune cells. An activated innate immune response is thought to contribute to chronic intrahepatic inflammation and to the response to IFN-based therapy. Natural killer (NK) cells are the most frequent innate immune cells in the liver, representing about 40% of the intrahepatic lymphocytic infiltrate. NK cells display increased pSTAT1mediated cytotoxicity and decreased pSTAT4-dependent IFN-&#947; production in patients with chronic HCV infection in comparison to those of uninfected control persons. This functional dichotomy is thought to be driven by chronic exposure to low levels of HCVinduced IFN-&#945;, which increases STAT1/pSTAT1 levels in NK cells. In the fist project we asked whether the immunomodulatory drug ribavirin alters NK cell responses. Ribavirin is an important component of interferon-based and direct antiviral treatment regimens for HCV infection. Immunomodulation, in particular improvement of the host interferon response, has been proposed as ribavirins mechanism of action. NK cells are sensitive biomarkers for IFN-&#945;/&#946; receptor signaling, as NK cell cytotoxicity and IFN-&#947; production are regulated by STAT1- and STAT4-phosphorylation, respectively. To assess whether ribavirin has a direct effect on NK cells and/or improves the IFN-&#947; response of NK cells in the presence of IFN-&#945;, we prospectively studied 22 HCV patients with and 32 patients without 4 weeks of ribavirin pretreatment, who all received subsequent PegIFN/ribavirin combination therapy. During ribavirin pretreatment, the frequency of CD56dim NK cells with cytotoxic effector functions decreased (p=0.049) as did the frequency of CD56bright NK cells with the capacity to produce IFN-&#947; (p=0.001). In vitro or in vivo exposure of NK cells to ribavirin improved the pSTAT4 (p<0.01) but not pSTAT1 response of NK cells to subsequent stimulation with IFN-&#945;. This was associated with an increase in IFN-&#947; production but not cytotoxicity of NK cells during subsequent IFN-&#945;-based therapy. In addition the frequency of IFN-&#947;-producing NK cells was greater in fast second-phase virological responders than in slow responders. Thus, ribavirin enhances the pSTAT4 and IFN-&#947; response of NK cells to IFN-&#945; stimulation. In the second project, we asked whether the rapid elimination of HCV with direct-acting antivirals normalizes NK cell function. Peripheral blood and intrahepatic NK cells were collected from 11 patients with chronic HCV genotype 1b infection before and during a 24-week treatment course with asunaprevir/daclatasvir therapy and studied by flow cytometry. NK cell responses were compared to blood NK cells from 8 healthy controls. Seven patients responded with undetectable viremia by week 3-8, whereas four experienced a virological breakthrough between weeks 4-12 of treatment (week 4 n=1, week 6 n=2 and week 12 n=1). Analysis of NK cell responses has been completed for the first 8 weeks of the treatment protocol, and will continue for the end-of-treatment (week 24) time point and the post-treatment time points. In addition, we will study two patients who have recently enrolled in the study.